Detection Extraction and Evaluation of Phage Depolymerase Enzyme against Shiga-toxigenic Escherichia coli Biofilms

Introduction:  Shiga-toxigenic Escherichia coli(STEC) is a major group of foodborne pathogens that can produce strong biofilms on foods, and food contact surfaces. These biofilms can be difficult to remove using conventional sanitizers, but could be disrupted using bacteriophage, targeting specific bacteria. These phages produce depolymerase enzymes, to degrade STEC biofilms, allowing the phage to kill the host bacterium.

Purpose:  To evaluate biofilm inhibiting activity of depolymerase enzyme extracted from STEC specific phage.

Methods:  Previously sequenced phages, isolated in the lab, were tested for the presence of depolymerase-enzyme-coding gene in STEC-O45 using homologous sequence alignment tool (BLAST), PCR and gel electrophoresis. Crude depolymerase-enzyme was extracted using phage-lysate, centrifugation, dialysis, PEG concentration, and ultracentrifugation. Crude extract was then tested for inhibition against STEC-O45, using spot-on-lawn assay. Additionally, its biofilm disrupting capabilities were examined by allowing STEC-O45 (7 log₁₀ CFU/ml) to form biofilms in micro-titer plates for 24 hrs. Plates were then treated with phage, phage-depolymerase or PBS-control for 16 hrs and stained with crystal-violet. Biofilm disruption was measured as a change in absorbance (A₅₉₅) and was visually analyzed with Scanning Electron Microscopy (SEM). Surviving bacterial population was also enumerated on tryptic soy agar.

Results:  Sequences homologous to phage-enzyme were used to design primers. The presence of enzyme-encoding sequence in the phage was confirmed by PCR-positive band at ~800bp. Crude phage-enzyme extract showed lytic activity against STEC-O45 on spot-on-lawn assay. Phage and phage-enzyme treated wells showed reduction in absorbance (0.705 to 0.918) of STEC biofilms, compared to control. A reduction of 1.0-1.1 logs in STEC population was observed with phage and phage-depolymerase treatments. SEM images showed an extracellular matrix surrounding the colonies in PBS-control. However, phage and phage-depolymerase treated cells showed rough surfaces with filament-like cell envelopes, indicating cellular damage.

Significance:  Bacteriophage and phage-depolymerase can be applied as bio-control agents in the food industry to control STEC biofilms.